THE ROLE OF PDIA3 IN VITAMIN D SIGNALING IN OSTEOBLASTS A Dissertation Presented to The Academic Faculty By Jiaxuan Chen In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Department of Biomedical Engineering Georgia Institute of Technology December 2012 THE ROLE OF PDIA3 IN VITAMIN D SIGNALING IN OSTEOBLASTS Thesis committee members: Dr. Barbara D. Boyan, Advisor Dr. Todd C. McDevitt Department of Biomedical Engineering Department of Biomedical Engineering Georgia Institute of Technology Georgia Institute of Technology Dr. Zvi Schwartz Dr. Kirill S. Lobachev Department of Biomedical Engineering School of Biology Georgia Institute of Technology Georgia Institute of Technology Dr. Hong Chen School of Medicine Emory University ACKNOWLEDGEMENTS First, I would like to thank my advisors Dr. Barbara D. Boyan and Dr. Zvi Schwartz. In 2007, I came from China to Georgia Tech to pursue my Ph.D. Dr.Boyan was kind enough to take a young international student with little related research experience. Through my five year period of Ph.D. study, Dr. Boyan has exposed me to many different perspectives of being a good researcher. I have learned how to properly interpret the data, critique a paper, make a presentation, give a talk, submit a manuscript, respond to reviewers, and many other skills. For this, I am very thankful. Besides the field of research, Dr.Boyan always gave me support to pursuit what is the best of my interests. I am very grateful for her and Dr.Schwartz’s support on my transferring my field of study to become a biomedical engineer, so I have the opportunity to establish my career in the field I am most interested in. Dr.Schwartz is the person who was involved in the design of every experiment conducted in this thesis. He has taught me how to make a hypothesis, how to design an experiment and how to set up proper controls when I was still an inexperienced graduate student. Ever since then, we have been going through both the good results and the bad ones and experiencing the exciting moments and the frustrating ones. He has always been inspiring me, challenging me and demanding more from me. Because of him, I was able to accomplish this thesis today, for which I am greatly appreciative. I would also like to thank my thesis committee members. Dr. Lobachev has been a great collaborator and has given me much help in establishing the site-directed mutations. Dr. McDevitt and Dr. Chen have provided their time and insight, and I am very appreciative to them for that. I would also like to thank the members of Boyan and Schwartz’s lab. They have been really helpful throughout the years. I would like to thank Leang Chhun, Crystal iii Branan, Sri Vemula and Lauren Carson for their help in cell culture. Ms. Brentis Henderson and Shannon Sullivan have made the administration process so much easier. Reyhaan Chaudhri, Sharon Hyzy, Dr. Rene Olivares-Navarrete and Dr. Yun Wang have taught me from how to use a pipette to many other useful lab techniques. Without their help, this study would not have been possible. All of the Boyan laboratory graduate students have made my life here more enjoyable: Jiaying Guo, Reyhaan Chaudhri, Bryan Bell, Tracy Denison, Chris Lee, Andrew Raines, Khairat El- Baradie, Jennifer Hurst-Kennedy, Jamie Lazin, Maya Fisher, Ming Zhong, Tanya Farooque, Chris Hermann, Rolando Gittens, Jennie Park, Maryam Doroudi, Brandy Rogers, Shirae Leslie, Meredith Myers, Qingfen Pan, Erin Hewitt, James Wade, Alice Cheung, and Srishti Bhutani. I would also like to thank my parents. Although they are oversea in China, they have always been there to support, motivate and comfort me. Without their help, I would not get to where I am today. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES .......................................................................................................... vii LIST OF FIGURES ....................................................................................................... viii SUMMARY ...................................................................................................................... x CHAPTER 1: Introduction ................................................................................................1 1.1 The significance of vitamin D research ................................................................ 1 1.2 Vitamin D Family ..................................................................................................2 1.3 The metabolic pathway of vitamin D .................................................................... 2 1.4 The function of 1α,25(OH)2D3 ............................................................................. 5 1.5 The genomic pathways and nuclear vitamin D receptor ....................................... 5 1.6 Rapid responses and plasma membrane vitamin D receptor ............................... 6 1.7 Thesis objective ...................................................................................................9 1.8 References ........................................................................................................ 11 CHAPTER 2: Pdia3 mediates the membrane response to 1α,25-Dihydroxyvitamin D3 in osteoblasts .................................................................................................................... 15 2.1 Introduction ........................................................................................................ 15 2.2 Materials and Methods ....................................................................................... 17 2.3 Results .............................................................................................................. 24 2.4 Discussion ......................................................................................................... 36 2.5 References ........................................................................................................ 42 CHAPTER 3: Plasma membrane Pdia3 and VDR interact to elicit rapid responses to 1α,25(OH)2D3 ............................................................................................................... 45 3.1 Introduction ........................................................................................................ 45 3.2 Materials and Methods ....................................................................................... 48 3.3 Results .............................................................................................................. 60 3.4 Discussion ......................................................................................................... 73 v 3.5 References ........................................................................................................ 80 CHAPTER 4. Chaperone properties of Pdia3 participate in rapid plasma membrane mediated actions of 1α, 25-dihydroxyvitamin D3 ............................................................ 83 4.1 Introduction ........................................................................................................ 83 4.2 Materials & Methods .......................................................................................... 87 4.3 Results .............................................................................................................. 97 4.4 Discussion ....................................................................................................... 108 4.5 References ...................................................................................................... 114 CHAPTER 5: Mineralization of three dimensional osteoblast cultures is enhanced by interaction of 1α,25-dihydroxyvitamin D3 and BMP2 via two specific vitamin D receptors .................................................................................................................................... 117 5.1 Introduction ...................................................................................................... 117 5.2 Materials & Methods ........................................................................................ 119 5.3 Results ............................................................................................................ 129 5.4 Discussion ....................................................................................................... 136 . 5.5 References ...................................................................................................... 143 CHAPTER 6: Summary ............................................................................................... 146 6.1 The role of Pdia3 in rapid responses to 1α,25(OH)2D3 in osteoblasts. ............ 146 6.2 The relative role of the two plasma membrane receptors of 1α,25(OH)2D3, Pdia3 and VDR .......................................................................................................... 146 6.3 The molecular mechanism of Pdia3 as plasma membrane receptor of 1α,25(OH)2D3 ................................................................................................. 147 6.4 The role of Pdia3 and VDR in 3D mineralization .............................................. 148 6.5 Conclusions ..................................................................................................... 149 6.6 Discussion and significance ............................................................................. 150 6.7 References .....................................................................................................